Microfluidic platforms for advanced risk assessments of nanomaterials.

In the past few years, promising efforts to utilize microfabrication-based technologies have laid the foundation for developing advanced, and importantly more physiologically-realistic, microfluidic methods for risk assessment of engineered nanomaterials (ENMs). In the present review, we discuss the wave of recent developments using microfluidic-based in vitro models and platforms for nanotoxicological assays, such as determination of cell viability, cellular dose, oxidative stress and nuclear damage. Here, we specifically highlight the tangible advantages of microfluidic devices in providing promising tools to tackle many of the current and ongoing challenges faced with traditional toxicology assays. Most importantly, microfluidic technology not only allows to recreate physiologically-relevant in vitro models for nanotoxicity examinations, but also provides platforms that deliver an attractive strategy towards improved control over applied ENM doses. In a final step, we present examples of state-of-the-art microfluidic platforms for in vitro assessment of potential adverse ENM effects.

Enhanced upconversion luminescence in NaGdF4:Yb,Er nanocrystals by Fe3+ doping and their application in bioimaging.

The visible green and red upconversion emissions in Er(3+)/Yb(3+) doped ß-NaGdF4 nanoparticles were enhanced by tridoping with Fe(3+) ions (0-40 mol%). XRD, XPS, ICP-AES and EDS data demonstrated successful incorporation of Fe(3+) ions in NaGdF4:Yb(3+)/Er(3+) nanoparticles. The effect of Fe(3+) tridoping on the upconversion luminescence in NaGdF4:Yb(3+)/Er(3+) NPs was investigated in detail. The green and red emission intensities were enhanced by 34 and 30 times, respectively. The maximum emission was observed in a sample containing 30 mol% Fe(3+) ions. A possible mechanism for the enhanced upconversion emission is proposed. In addition, a layer of silica was coated onto the surface of UCNPs to improve the biocompatibility. Folic acid was covalently linked to the silica coated UCNPs to form UCNP@SiO2-FA nanoprobes, which have been successfully applied to the fluorescent imaging HeLa cells.

Multiparametric assessment of Cd²+ cytotoxicity using MTT-based microfluidic image cytometry.

A modified MTT protocol-based microfluidic image cytometry (µFIC) was performed to assess Cd(2+) induced cytotoxicity. The expanded capabilities of µFIC, such as in situ measurement, high-throughput, and multiparametric analysis of adherent cells under precisely controlled chemical environments of microfluidic channels, were demonstrated in this study. Multiparametric analysis of µFIC data has enabled us to categorize the progress of cell death into at least four different subgroups based on their morphology and metabolic activity. These advantages of the MTT-based µFIC as a simpler, cheaper, and faster in vitro cell-based assay tool have many implications in biomedical, pharmaceutical, toxicological, and biological application areas, and we propose this technique as a future high throughput-high content screening (HT-HCS) platform for cytotoxicity assays and drug screening.

Integrated microfluidics platforms for investigating injury and regeneration of CNS axons.

We describe the development of experimental platforms to quantify the regeneration of injured central nervous system (CNS) neurons by combining engineering technologies and primary neuronal cultures. Although the regeneration of CNS neurons is an important area of research, there are no currently available methods to screen for drugs. Conventional tissue culture based on Petri dish does not provide controlled microenvironment for the neurons and only provide qualitative information. In this review, we introduced the recent advances to generate in vitro model system that is capable of mimicking the niche of CNS injury and regeneration and also of testing candidate drugs. We reconstructed the microenvironment of the regeneration of CNS neurons after injury to provide as in vivo like model system where the soluble and surface bounded inhibitors for regeneration are presented in physiologically relevant manner using microfluidics and surface patterning methods. The ability to control factors and also to monitor them using live cell imaging allowed us to develop quantitative assays that can be used to compare various drug candidates and also to understand the basic mechanism behind nerve regeneration after injury.

Synthesis and evaluation of cytotoxic effects of hanultarin and its derivatives.

One of the known cytotoxic lignans is (-)-1-O-feruloyl-secoisolariciresinol designated as hanultarin, which was isolated from the seeds of Trichosanthes kirilowii. In this Letter, we described the first synthesis of 1-O-feruloyl-secoisolariciresinol, 1,4-O-diferuloyl-secoisolariceresinol and their analogues. The cytotoxicities of these compounds were evaluated against several cancer cell lines. Interestingly, we found that the feruloyl diester derivative of secoisolariciresinol was the most active cytotoxic compound against all the cancer cells tested in this experiment. The IC(50) values of the1,4-O-diferuloyl-secoisolariceresinol were in the range of 7.1-9.8µM except one cell line. In considering that both ferulic acid and secoisolariciresinol are commonly found in many plants and have no cytotoxicity, this finding is remarkable in that simple covalent bonds between the ferulic acid and secoisolariciresinol cause a cytotoxic effect.

A new perspective on in vitro assessment method for evaluating quantum dot toxicity by using microfluidics technology.

In this study, we demonstrate a new perspective on in vitro assessment method for evaluating quantum dot (QD) toxicity by using microfluidics technology. A new biomimetic approach, based on the flow exposure condition, was applied in order to characterize the cytotoxic potential of QD. In addition, the outcomes obtained from the flow exposure condition were compared to those of the static exposure condition. An in vitro cell array system was established that used an integrated multicompartmented microfluidic device to develop a sensitive flow exposure condition. QDs modified with cetyltrimethyl ammonium bromide∕trioctylphosphine oxide were used for the cytotoxicity assessment. The results suggested noticeable differences in the number of detached and deformed cells and the viability percentages between two different exposure conditions. The intracellular production of reactive oxygen species and release of cadmium were found to be the possible causes of QD-induced cytotoxicity, irrespective of the types of exposure condition. In contrast to the static exposure, the flow exposure apparently avoided the gravitational settling of particles and probably assisted in the homogeneous distribution of nanoparticles in the culture medium during exposure time. Moreover, the flow exposure condition resembled in vivo physiological conditions very closely, and thus, the flow exposure condition can offer potential advantages for nanotoxicity research.

Assessment of cytocompatibility of surface-modified CdSe/ZnSe quantum dots for BALB/3T3 fibroblast cells.

With the widespread use of quantum dots (QDs), the likelihood of exposure to QDs has been assumed to have increased substantially. Recently, QDs have been employed in numerous biological and medical applications. However, there is a lack of toxicological data pertaining to QDs. In this study, we aimed to investigate the cytocompatibility of surface-modified CdSe/ZnSe QDs for BALB/3T3 fibroblast cells. The ligands used for surface modification are mercaptopropionic acid (MPA) and Gum arabic (GA)/tri-n-octylphosphine oxide (TOPO). Cells were exposed to different concentrations of QDs followed by illustrative cytotoxicity analyses. Furthermore, we used a confocal microscope to assess intracellular uptake of QDs. Confocal images showed that MPA-coated QDs were distributed inside the cytoplasmic region of cells. In contrast, GA/TOPO-coated QDs were not found inside cells. MPA-coated QDs were highly cytocompatible, whereas GA/TOPO-coated QDs were toxic to the cells. Cells treated with GA/TOPO-coated QDs showed altered morphology, decreased viability, significant concentrations of intracellular free cadmium, detectable reactive oxygen species (ROS) formation, depolymerized cytoskeleton, and irregular-shaped nuclei. This study suggests that surface modification by ligands plays a significant role in the prevention of cytotoxicity of QDs.

Morphology-based assessment of Cd2+ cytotoxicity using microfluidic image cytometry (microFIC).

Microfluidic systems have significant implications in the field of in vitro cell-based assays since they may allow conventional cell-based assays to be conducted in an automated and high-throughput fashion. In this study, we combined a simple microfluidic cells-on-chip system with a morphology-based image cytometric analysis approach for the assessment of Cd(2+) induced apoptosis of Chang liver cell line. A simple and efficient in situ monitoring method for quantifying the progress of a cell death event was developed and is presented here. Reasonable agreement of the estimated EC(50) value from this study with those from the literature and a close correlation between the observed changes in cell morphology (i.e., circularity) and the amount of reactive oxygen species (ROS) generation confirmed the validity of this morphology-based microfluidic image cytometric (microFIC) assessment method. We propose this morphology-based microFIC approach as an easy and efficient way to assess cytotoxicity which can be adapted to high-throughput screening platforms for in vitro cytotoxicity assays as well as drug screening.

Multicompartmented microfluidic device for characterization of dose-dependent cadmium cytotoxicity in BALB/3T3 fibroblast cells.

This paper describes the development of a miniaturized multicompartmented microfluidic device for high-throughput cell cytotoxicity assays and its applicability to the investigation of cadmium-induced cytotoxicity. A steady gradient of cadmium was generated inside the compartments to study the effects of cadmium ion on BALB/3T3 fibroblast cells in a dose-dependent fashion. The device allowed the performance of multiplexed assays to probe the dosage effect of cadmium, morphological alterations of live cells, regulation of proliferation and viability of cells, determination of reactive oxygen species, mechanisms of cell death, i.e. apoptosis and/or necrosis, and immunocytochemical staining of cells in parallel and/or serially, or on a single population simultaneously. The outcomes of all the microfluidic assays were compared to conventional plates-based cytotoxicity assays. The results indicated that the cells cultured in this device were morphologically healthy with greater than 90% viability. They further suggested that the basic mode of cell death behind cadmium-induced cytotoxicity was apoptosis, which was regulated by intracellular oxidative stress via cytoskeleton disorganization and nuclear condensation. Such microenvironments resemble the in vivo physiological conditions very closely and thus offer a unique platform for more accurate observations of cytotoxicity assays and more precise estimation of the IC(50) value in comparison to conventional analytical assays.

Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber.

We have developed a simple microfluidic device for generating stable concentration gradients in 2D and 3D environments. The device, termed the Ladder Chamber, uses a two-compartment diffusion system to generate steady state gradients across flow-free channels that connect the source and sink channels. To demonstrate the utility of the Ladder Chamber for cell migration, neutrophil chemotaxis was successfully observed in soluble chemoattractant (IL-8) gradient. The Ladder Chamber's simple design and experimental implementation make it an attractive approach for investigating cell migration and other biological experiments in well-defined gradients in 2D surfaces as well as in 3D gels.

External force-assisted cell positioning inside microfluidic devices.

This paper describes straightforward approaches to positioning cells within microfluidic devices that can be implemented without special equipment or fabrication steps. External forces can effectively transport and position cells in preferred locations inside microfluidic channels. Except for centrifugal force-based positioning that can be used with any microfluidic channels, hydrodynamic and gravitational force-based positioning yield reproducible and biocompatible results when implemented with a microfluidic "module" that contains a barrier with embedded microgrooves. Primary rat cortical neurons, metastatic human breast cancer cells MDA-MB-231, NIH 3T3 mouse fibroblasts, and human umbilical vein endothelial cells (HUVECs) were compatible with the positioning processes. After positioning, cells attached, proliferated and migrated like control cells that were cultured on tissue culture dishes or glass coverslips. No apparent morphological differences were observed in positioned cells compared with control cells. Finally, to demonstrate a practical application of the methods, cells were placed in a single row along a wall inside a microfluidic chemotaxis chamber (MCC), and were exposed to stable concentration gradient of chemoattractant. Cell positioning allows that all cells get exposed to the same level of chemoattractant at the start of the experiment helping standardize cellular response.

Microfluidic chambers for cell migration and neuroscience research.

This chapter describes the fabrication and use microfluidic chambers for cell migration and neuroscience research. Both microfluidic chambers are made using soft lithography and replica molding. The main advantages of using soft lithography to create microfluidic chambers are reproducibility, ease of use, and straightforward fabrication procedures. The devices can be fabricated in biology and chemistry laboratories with minimal access to clean-room facilities. First, a microfluidic chemotaxis chamber, which has been used in investigating chemotaxis of neutrophils, human breast cancer cells, and other cell types, is described. Precise and stable gradients of chemoattractants with arbitrary shapes can be generated for different applications. Second, a multicompartment culture chamber that can fluidically isolate neuronal processes from cell bodies is described. The design of this chamber is such that only neurites grow through a series of microgrooves embedded in a physical barrier. Both devices are compatible with phase, differential interference contrast, and fluorescence microscopy.

Microfluidic culture platform for neuroscience research.

This protocol describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) and peripheral nervous system neurons for neuroscience applications. This method uses replica-molded transparent polymer parts to create miniature multi-compartment cell culture platforms. The compartments are made of tiny channels with dimensions of tens to hundreds of micrometers that are large enough to culture a few thousand cells in well-controlled microenvironments. The compartments for axon and somata are separated by a physical partition that has a number of embedded micrometer-sized grooves. After 3-4 days in vitro (DIV), cells that are plated into the somal compartment have axons that extend across the barrier through the microgrooves. The culture platform is compatible with microscopy methods such as phase contrast, differential interference microscopy, fluorescence and confocal microscopy. Cells can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration. This protocol can be completed in 1-2 days.

A microfluidic culture platform for CNS axonal injury, regeneration and transport.

Investigation of axonal biology in the central nervous system (CNS) is hindered by a lack of an appropriate in vitro method to probe axons independently from cell bodies. Here we describe a microfluidic culture platform that polarizes the growth of CNS axons into a fluidically isolated environment without the use of targeting neurotrophins. In addition to its compatibility with live cell imaging, the platform can be used to (i) isolate CNS axons without somata or dendrites, facilitating biochemical analyses of pure axonal fractions and (ii) localize physical and chemical treatments to axons or somata. We report the first evidence that presynaptic (Syp) but not postsynaptic (Camk2a) mRNA is localized to developing rat cortical and hippocampal axons. The platform also serves as a straightforward, reproducible method to model CNS axonal injury and regeneration. The results presented here demonstrate several experimental paradigms using the microfluidic platform, which can greatly facilitate future studies in axonal biology.

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device.

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.

Patterned cell culture inside microfluidic devices.

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.

Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.

This paper describes a microfluidic approach to generate dynamic temporal and spatial concentration gradients using a single microfluidic device. Compared to a previously described method that produced a single fixed gradient shape for each device, this approach combines a simple "mixer module" with gradient generating network to control and manipulate a number of different gradient shapes. The gradient profile is determined by the configuration of fluidic inputs as well as the design of microchannel network. By controlling the relative flow rates of the fluidic inputs using separate syringe pumps, the resulting composition of the inlets that feed the gradient generator can be dynamically controlled to generate temporal and spatial gradients. To demonstrate the concept and illustrate this approach, examples of devices that generate (1) temporal gradients of homogeneous concentrations, (2) linear gradients with dynamically controlled slope, baseline, and direction, and (3) nonlinear gradients with controlled nonlinearity are shown and their limitations are described.

Microfluidic Multicompartment Device for Neuroscience Research.

This paper describes and characterizes a novel microfabricated neuronal culture device. This device combines microfabrication, microfluidic, and surface micropatterning techniques to create a multicompartment neuronal culturing device that can be used in a number of neuroscience research applications. The device is fabricated in poly(dimethylsiloxane), PDMS, using soft lithography techniques. The PDMS device is placed on a tissue culture dish (polystyrene) or glass substrate, forming two compartments with volumes of less than 2 µL each. These two compartments are separated by a physical barrier in which a number of micron-size grooves are embedded to allow growth of neurites across the compartments while maintaining fluidic isolation. Cells are plated into the somal (cell body) compartment, and after 3-4 days, neurites extend into the neuritic compartment via the grooves. Viability of the neurons in the devices is between 50 and 70% after 7 days in culture; this is slightly lower than but comparable to values for a control grown on tissue culture dishes. Healthy neuron morphology is evident in both the devices and controls. We demonstrate the ability to use hydrostatic pressure to isolate insults to one compartment and, thus, expose localized areas of neurons to insults applied in soluble form. Due to the high resistance of the microgrooves for fluid transport, insults are contained in the neuritic compartment without appreciable leakage into the somal compartment for over 15 h. Finally, we demonstrate the use of polylysine patterning in combination with the microfabricated device to facilitate identification and visualization of neurons. The ability to direct sites of neuronal attachment and orientation of neurite outgrowth by micropatterning techniques, combined with fluidically isolated compartments within the culture area, offers significant advantages over standard open culture methods and other conventional methods for manipulating distinct neuronal microenvironments.